Bottom Line:
Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters.Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters.This process does not require additional determinants located in the ectodomain.

ABSTRACTMuscle-specific receptor tyrosine kinase (MuSK) is required for the formation of the neuromuscular junction. Using direct gene transfer into single fibers, MuSK was expressed extrasynaptically in innervated rat muscle in vivo to identify its contribution to synapse formation. Spontaneous MuSK kinase activity leads, in the absence of its putative ligand neural agrin, to the appearance of epsilon-subunit-specific transcripts, the formation of acetylcholine receptor clusters, and acetylcholinesterase aggregates. Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters. The contribution of endogenous MuSK was excluded by using genetically altered mice, where the kinase domain of the MuSK gene was flanked by loxP sequences and could be deleted upon expression of Cre recombinase. This allowed the conditional inactivation of endogenous MuSK in single muscle fibers and prevented the induction of ectopic AChR clusters. Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters. This process does not require additional determinants located in the ectodomain.

fig1: MuSK induces postsynaptic-like specializations. (A) Plasmid DNA for nGFP and MuSK was injected ectopically in single muscle fibers of innervated soleus muscles of rats. Muscles were excised 21 d after injection. Green fluorescence of nGFP in single fibers indicated expression of transgenic proteins (unpublished data). AChR clusters induced by MuSK are detected by staining with r-bgt (7 rats were used for DNA injection; a total of 96 fibers were injected and AChR clusters were detected in 61 fibers). (B) Muscle fibers were injected with plasmid DNA for nGFP, MuSK, and rapsyn and analyzed as in A. Expression of MuSK and rapsyn induces AChR clusters comparable to clusters observed in A. 10 rats were used for DNA injection; a total of 138 fibers were injected and AChR clusters were detected in 76 fibers. A and B are projections of confocal images. (C–F) Muscle fibers were injected with plasmid DNA for nGFP, MuSK, and rapsyn. Muscles were excised 28 d after injection and analyzed as in A. (C) Fluorescence microscopic view of r-bgt–labeled AChR, induced by transgenic MuSK. (D) AChE aggregates, visualized according to Koelle and Friedenwald (1949), appear colocalized with the MuSK-induced AChR clusters (3 rats were used for DNA injection and a total of 59 fibers were injected. AChR clusters were detected together with AChE aggregates on 34 fibers). In control-injected fibers, no AChE clusters were detected (3 rats were used for nGFP DNA injection without MuSK and rapsyn DNA; a total of 66 fibers were injected and 31 fibers were nGFP-positive but without AChR or AChE clusters). (E) Cross-section of muscle region containing fiber with MuSK-induced ectopic AChR clusters. AChR clusters are located in the plasma membrane of the injected muscle fiber. (F) Cross-section, shown in E, was subjected to in situ hybridization using an ε subunit–specific, 35S-labeled antisense probe. No hybridization signals were detected using sense probes for hybridization (unpublished data). The AChR-expressing fiber contains ε subunit–specific mRNA (three independent hybridization experiments were performed with thin sections of soleus muscle from two rats; AChR-positive fibers expressed in all cases ε-subunit transcripts).

Mentions:
By direct gene transfer into individual muscle fibers it was shown that agrin induced the formation of ectopic AChR clusters (Cohen et al., 1997; Jones et al., 1997; Meier et al., 1997; Rimer et al., 1997). Using the same approach, it was found that injection of MuSK plasmid DNA alone results in the absence of neural agrin and also in the formation of AChR clusters (Fig. 1 A; Hesser et al., 1999; Jones et al., 1999). MuSK-induced AChR clusters differed, however, from clusters induced following overexpression of neural agrin. Agrin-induced clusters appeared larger in size and more spread out both on the injected fiber as well as on adjacent fibers (Jones et al., 1997; see Figs. 3 and 4). In the case of rapsyn, which is essential for the clustering of AChR at developing synapses (Gautam et al., 1995), we observed that ectopic injection of rapsyn plasmid DNA alone did not induce AChR clustering (unpublished data). Since rapsyn could be linked to MuSK and AChR (Apel et al., 1997), we asked whether MuSK-induced AChR clusters might be altered when expressed in the presence of transgenic rapsyn. Coinjection of MuSK and rapsyn DNA resulted in the formation of AChR clusters (Fig. 1 B) which were similar to the clusters induced by MuSK alone as shown in Fig. 1 A.

fig1: MuSK induces postsynaptic-like specializations. (A) Plasmid DNA for nGFP and MuSK was injected ectopically in single muscle fibers of innervated soleus muscles of rats. Muscles were excised 21 d after injection. Green fluorescence of nGFP in single fibers indicated expression of transgenic proteins (unpublished data). AChR clusters induced by MuSK are detected by staining with r-bgt (7 rats were used for DNA injection; a total of 96 fibers were injected and AChR clusters were detected in 61 fibers). (B) Muscle fibers were injected with plasmid DNA for nGFP, MuSK, and rapsyn and analyzed as in A. Expression of MuSK and rapsyn induces AChR clusters comparable to clusters observed in A. 10 rats were used for DNA injection; a total of 138 fibers were injected and AChR clusters were detected in 76 fibers. A and B are projections of confocal images. (C–F) Muscle fibers were injected with plasmid DNA for nGFP, MuSK, and rapsyn. Muscles were excised 28 d after injection and analyzed as in A. (C) Fluorescence microscopic view of r-bgt–labeled AChR, induced by transgenic MuSK. (D) AChE aggregates, visualized according to Koelle and Friedenwald (1949), appear colocalized with the MuSK-induced AChR clusters (3 rats were used for DNA injection and a total of 59 fibers were injected. AChR clusters were detected together with AChE aggregates on 34 fibers). In control-injected fibers, no AChE clusters were detected (3 rats were used for nGFP DNA injection without MuSK and rapsyn DNA; a total of 66 fibers were injected and 31 fibers were nGFP-positive but without AChR or AChE clusters). (E) Cross-section of muscle region containing fiber with MuSK-induced ectopic AChR clusters. AChR clusters are located in the plasma membrane of the injected muscle fiber. (F) Cross-section, shown in E, was subjected to in situ hybridization using an ε subunit–specific, 35S-labeled antisense probe. No hybridization signals were detected using sense probes for hybridization (unpublished data). The AChR-expressing fiber contains ε subunit–specific mRNA (three independent hybridization experiments were performed with thin sections of soleus muscle from two rats; AChR-positive fibers expressed in all cases ε-subunit transcripts).

Mentions:
By direct gene transfer into individual muscle fibers it was shown that agrin induced the formation of ectopic AChR clusters (Cohen et al., 1997; Jones et al., 1997; Meier et al., 1997; Rimer et al., 1997). Using the same approach, it was found that injection of MuSK plasmid DNA alone results in the absence of neural agrin and also in the formation of AChR clusters (Fig. 1 A; Hesser et al., 1999; Jones et al., 1999). MuSK-induced AChR clusters differed, however, from clusters induced following overexpression of neural agrin. Agrin-induced clusters appeared larger in size and more spread out both on the injected fiber as well as on adjacent fibers (Jones et al., 1997; see Figs. 3 and 4). In the case of rapsyn, which is essential for the clustering of AChR at developing synapses (Gautam et al., 1995), we observed that ectopic injection of rapsyn plasmid DNA alone did not induce AChR clustering (unpublished data). Since rapsyn could be linked to MuSK and AChR (Apel et al., 1997), we asked whether MuSK-induced AChR clusters might be altered when expressed in the presence of transgenic rapsyn. Coinjection of MuSK and rapsyn DNA resulted in the formation of AChR clusters (Fig. 1 B) which were similar to the clusters induced by MuSK alone as shown in Fig. 1 A.

Bottom Line:
Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters.Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters.This process does not require additional determinants located in the ectodomain.

ABSTRACTMuscle-specific receptor tyrosine kinase (MuSK) is required for the formation of the neuromuscular junction. Using direct gene transfer into single fibers, MuSK was expressed extrasynaptically in innervated rat muscle in vivo to identify its contribution to synapse formation. Spontaneous MuSK kinase activity leads, in the absence of its putative ligand neural agrin, to the appearance of epsilon-subunit-specific transcripts, the formation of acetylcholine receptor clusters, and acetylcholinesterase aggregates. Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters. The contribution of endogenous MuSK was excluded by using genetically altered mice, where the kinase domain of the MuSK gene was flanked by loxP sequences and could be deleted upon expression of Cre recombinase. This allowed the conditional inactivation of endogenous MuSK in single muscle fibers and prevented the induction of ectopic AChR clusters. Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters. This process does not require additional determinants located in the ectodomain.